Using microarray and real time RT-PCR analysis to study the molecular changes associated with this thymic loss, we have examined the transcriptomes of the thymi of male and female mice at various stages of thymic aging and fed under caloric restricted (CR) or ad libitum (AL) diets. Upon identification of a pool of putative genes associated with thymic aging by microarrays, quantitative RT-PCR was utilized to validate and examine the role of contributing biological variables. Genes involved in various biological and molecular processes including cell-cycle progression, transcriptional regulators, maintenance and remodeling of extracellular matrix, protein binding and transport, proteasomal proteins, apoptosis, stress response, inflammation and immune function were observed to significantly change with thymic aging. However, the majority of differences among samples also depended on variables such as sex and diet, and not on age alone. Interestingly, many of the gene expression changes associated with thymic aging are either muted or almost completely reversed in mice placed on caloric restricted diets. These studies provide valuable insight into the molecular mechanisms associated with thymic aging and the potential aging prevention effects of dietary restriction. Moreover, these results emphasize the need to account for biological variables such as sex and dietary interactions to elucidate the genomic correlates that influence the molecular pathways responsible for thymic involution. We are also currently completing the analysis and confirmation of these data from mice of various ages infused with various hormones that result in a partial restoration in thymocyte numbers. Array analysis of the thymi of such treated mice may yield valuable data on the common molecular processes involved in thymic regeneration. It is unclear whether certain lymphoid organs or cellular components play a critical role in longevity and lifespan. The overall goal of this project is to produce a comprehensive gene expression profile in the thymus, spleen, and lymph nodes during the aging process to identify unique and common genes and functionally related groups of genes that are expressed in age-dependent manner in these different organ systems. We have initially focused our efforts on the thymus, as its involution is believed to be one of the most significant obstacles to overcome in addressing the immunological deficits associated with aging.